The most common cause of bloody diarrhea and hemolytic uremic syndrome (HUS) in North America is infection by enterohemorrhagic E. coli (EHEC) (1). Alternative names for EHEC are Shiga toxin-producing E. coli (STEC), Shiga-like toxin-producing E. coli (SLTEC), Verocytotoxin-producing E. coli (VTEC), or Verotoxin-producing E. coli. In the United States, this food-borne E. coli is the most common infectious cause of bloody diarrhea in individuals of all ages. HUS is the most common cause of kidney failure in children in the U.S. and Canada.
This organism was the cause of the infamous "Jack-in-the-Box" food-poisoning outbreak in Seattle in 1993 which infected over 500 people and resulted in 4 deaths and many cases of long-term kidney damage. In 1996, this organism caused an enormous outbreak involving more than 8,000 people in Japan, resulting in 7 deaths. In late 1996, EHEC again caused an outbreak of food-poisoning in Scotland which affected 250 people and killed 18 people.
The most important virulence factor of E. coli associated with HUS is a potent cytotoxin known as Shiga toxin, Shiga-like toxin, verocytotoxin, or verotoxin, also called Stx. After production by E. coli colonizing the large intestine, Shiga toxin is absorbed into the circulation and eventually affects the kidney. The evidence linking Stx to HUS is both epidemiological and experimental. The first epidemiological association between Stx-producing E. coli and HUS was made by Karmali et al. (2) in 1983, and numerous studies since then have supported this association. Subsequent in vitro studies have shown that purified Stx has profound effects on renal endothelial cells resulting in cell death (3).
However, besides the clear-cut involvement of Stx in pathogenesis, little is known about other bacterial virulence factors involved in this disease. The lipopolysaccharide (LPS) of EHEC has been reported to enhance the effect of the Stx on human vascular endothelial cells (4), although the exact mechanism is not known. The other virulence factor of this organism that has been implicated in animal models is the 94 kilodalton outer membrane protein (OMP) known as intimin, which was discovered in the inventor's laboratory (5, 6). Intimin is involved in the colonization of the intestinal tract, which is apparently necessary for disease, but there is no evidence that intimin is directly involved in the renal disease. Since oral ingestion of preformed toxin is apparently not sufficient for causing HUS, other bacterial virulence factors clearly must be involved in the pathogenesis of this disease.
Two distinct sites of Disease, Intestinal and Renal
Disease due to EHEC starts by ingestion of meat, water, or other items that are contaminated with this organism. The organism then colonizes the large bowel where it can produce non-bloody diarrhea or bloody diarrhea (hemorrhagic colitis). In the colon, EHEC produces mucosal edema, erythema, ulceration and hemorrhage. A characteristic histopathology known as attaching and effacing (AE) results which is characterized by effacement of intestinal microvilli, intimate adherence of bacteria to enterocytes, and accumulation of polymerized actin and other cytoskeletal components in the epithelial cell directly beneath the adherent bacteria. The AE lesion has been repeatedly demonstrated in animals infected with EHEC and in cultured human epithelial cells. AE is assumed to occur in the colon early during the course of human infection, although direct evidence is lacking, probably because patients with EHEC infections undergo colonoscopy relatively late in the infection when the colonic surface has been denuded of epithelial cells. The AE histopathology is similar to that seen with enteropathogenic E. coli (EPEC), which do not produce Stx. We have shown that formation of the AE lesion by both EPEC and EHEC is mediated by the gene products of a 35 kilobase region of chromosomal DNA present in these strains but absent from normal flora E. coli (5, 7).
The pathogenic mechanisms by which EHEC produces non-bloody diarrhea and bloody diarrhea (hemorrhagic colitis) are largely unknown. Formation of the AE lesion in the absence of Stx is believed to be sufficient to cause non-bloody diarrhea in EPEC infections. Pure Stx can act as an enterotoxin, and in studies using rabbit jejunal tissue mounted in Ussing chambers, Stx selectively kills the absorptive tip cells while not affecting the secretory crypt cells, thereby changing the net balance of secretory/absorptive processes towards secretion (8). The bloody diarrhea is presumably due at least in part to the powerful cytotoxic effects of Stx, although the contribution of other bacterial factors and the host inflammatory response is not known. In at least two animal systems, gnotobiotic piglets and rabbits, Stx was not required for EHEC strains to alter secretory activity or cause severe histological changes (9, 10). O'Loughlin and colleagues (9, 11) have shown that disruption of colonic epithelium and changes in electrolyte transport during EHEC infection in rabbits are mediated by the host inflammatory response and that bacterial products other than Stx and factors encoded on the 60 MDa plasmid (see below) are necessary for the intestinal manifestations of EHEC disease.
The classic HUS triad includes microangiopathic hemolytic anemia, thrombocytopenia, and renal failure and may be accompanied by central nervous system manifestations in 30-50% of patients (12). Although hemolytic uremic syndrome (HUS) occurs in only 2-7% of all EHEC infections (while bloody diarrhea occurs in 90% of all infections (1,13)), HUS is associated with the greatest mortality due to this organism. The Shiga toxin produced in the bowel reaches the circulation and produces vascular endothelial damage that results in occlusion of the renal glomerular microvasculature by fibrin and platelets. Induction of inflammatory cytokines has also been suggested to contribute to the disease process (14-16). Although the preeminence of Stx in the disease process is accepted, the role of other bacterial factors in producing host damage or facilitating the delivery of Stx is unknown.
Virulence Factors of E. coli O157:H7
The majority of work on pathogenic factors of EHEC has focused on the Shiga toxins, which are encoded on bacteriophage inserted into the chromosome. Additional potential virulence factors are encoded in the chromosome and on a 60 MDa plasmid found in most strains of EHEC
Toxins Stx occurs in two major forms, stx1 and Stx2, which share 55 and 57% sequence identity in the A and B subunits, respectively (17). While stx1 is highly conserved, sequence variation exists within Stx2. The toxins consist of a single A subunit of ca. 32 kDa and 5 identical B subunits of ca. 7.7 kDa (18) The B subunit serves to bind the toxin to a specific glycolipid receptor, globotriaosylceramide or Gb.sub.3, while the A subunit is internalized and cleaves N-glycoside bonds on the 28S rRNA of the 60S ribosome. The resulting disruption of protein synthesis leads to death of renal endothelial cells, intestinal epithelial cells, vero or Hela cells, or any cell which possesses the Gb.sub.3 receptor.
The 60 MDa plasmid commonly found in EHEC strains contains genes encoding an .alpha.-hemolysin (19). Although this hemolysin is widely distributed among Stx-producing strains of E. coli, there are no data indicating that it is expressed in vivo or involved in pathogenesis of disease. Two other distinctly different phage-encoded hemolysins, termed enterohemolysins, are produced by many Stx-producing E. coli (20, 21) but again, there are no data to suggest in vivo expression or any role in pathogenesis.
Intestinal Adherence Factors
The only potential EHEC adherence factor which has been demonstrated to play a role in intestinal colonization in vivo in an animal model is the outer membrane protein intimin, encoded by the eaeA gene, also known as eae. We prepared an isogenic derivative of an EHEC strain specifically mutated in eaeA. In both conventional and gnotobiotic piglets (5, 22), a functional intimin protein was necessary for intimate adherence to intestinal epithelial cells, formation of the AE lesion, and induction of diarrhea. The importance of intimin in these processes was independently confirmed by other investigators using a different EHEC eaeA mutant in gnotobiotic piglets (23).
Other candidate adhesins have been reported but none have been well characterized or specifically demonstrated to play a role in adherence in vivo. Sherman et al. (24) reported that a 94 kDa OMP distinct from intimin (25) mediated adherence to Hep-2 epithelial cells, but no further characterization of this factor has been reported. Strains of EHEC produce fimbriae which might aid intestinal adherence (26-29), but no purified fimbriae or cloned fimbriae genes have been reported. An initial report (27) suggested that the 60 MDa plasmid was required for expression of fimbriae and adhesion to epithelial cells, but subsequent studies have reported that loss of the plasmid either enhanced adhesion (28), decreased adhesion (24), or had no effect on adhesion (29). The potential role of lipopolysaccharide (LPS) in adhesion was examined, and loss of LPS actually increased adherence to cultured epithelial cells (31). The existence of intestinal adherence factors distinct from intimin is suggested by the isolation of Stx-producing E. coli strains of serotypes other than EHEC that lack the eaeA gene but are still associated with bloody diarrhea or HUS in humans.
Other Potential Factors
LPS may either enhance or inhibit the toxicity of Stx in animal models (32,33) and enhances the cytotoxicity of Stx on human vascular endothelial cells in vitro (4). However, this effect is not specific for EHEC LPS since LPS from several species of the Enterobacteriaceae have similar effects (4). There is one report (34) that EHEC can invade cultured intestinal cell lines, but a later report (35) disputed these findings, showing that EHEC strains were no more invasive than normal flora E. coli. Furthermore, there is no in vivo evidence that invasion occurs in humans or in animals.
Non-0157:H7 STEC
Enterohemorrhagic E. coli O157:H7 is the most important type of E. coli that can cause HUS and bloody diarrhea. The O157:H7 nomenclature refers to particular bacterial surface antigens that define a serotype. The "O" designation refers to the surface lipopolysaccharide and the "H" antigen refers to the bacterial flagellar protein. Those E. coli that have the O157 and H7 antigens have the full array of virulence factors (Stx and intimin) and are always considered to be pathogens (36). However, there are similar E. coli that do not have the O157:H7 serotype but can cause similar disease. These E. coli are of serotypes such as O26:H11 and are called non-O157:H7 STEC (or non-O157:H7 EHEC). The major potential virulence factor that these organisms share is the Stx toxin but not all non-O157:H7 STEC are pathogenic (36). Mere expression of Stx alone does not define a pathogen. In fact, up to 63% of meat samples in U.S. supermarkets contain non-O157:H7 STEC (36). In contrast, E. coli O157:H7 is rarely found in foods. When non-O157:H7 STEC are isolated from patients, it is considered a pathogen but when they are isolated from food or animals, their significance is unknown (36). The invention described herein aids the discrimination of non-O157:H7 STEC that are of public health significance from those that are of little or no public health significance.
The reservoir of E. coli O157:H7 and non-O157:H7 STEC is believed to be in animals, particularly cattle (1). However, O157:H7 is rarely isolated from cattle, usually from less than 1% of cattle in most surveys (1). In contrast, non-O157:H7 STEC can be isolated from up to 40% of cattle (1). One important difference between non-O157:H7 STEC that are isolated from cattle and non-O157:H7 STEC isolated from humans is possession of the eae gene. The majority of these strains that are isolated from human disease possess the eae gene (37) whereas only a minority of these strains that are isolated from cattle possess eae and are presumably pathogenic for humans. Possession of the eae gene correlates with possession of a large block of virulence genes called the LEE (7) that also encode secreted proteins that are the basis of the present invention.
Serologic R
Determining the complete immune response to an infecting agent has many applications for understanding and controlling an infectious disease. First, such information can be used in serdiagnostics and seroepidemiology studies to detect evidence of an infection where the actual infectious agent cannot be detected either because of the lack of an appropriate specimen or lack of sufficiently sensitive detection methods. Second, knowledge of the components of an infectious agent which engender an immune response can be directly applied to developing a vaccine or immunotherapeutic agent (e.g., passive immunoglobulin). Third, detection of an immune response to a particular component of the infectious agent is direct evidence that this component is actually expressed in vivo during the course of infection, supporting the possibility that it plays a role in the pathogenesis of disease. The conclusion that an immune response indicates in vivo expression of the antigen is not always valid if, for example, the antigen is a toxin that is preformed at the time of ingestion or the infectious agent directly enters the blood stream without replication. However, for an agent that is ingested and replicates in the intestine, an immune response to a component strongly implies in vivo expression. For these reasons, a fuller determination of the immune response to E. coli O157:H7 is important for a better understanding of this disease.
Previous studies of the human immune response to E. coli O157:H7 have focused on the serological response since there is no evidence that cell-mediated immunity plays a role in this disease. The most widely studied antigen is the LPS. Chart et al. (38) showed that in one study of 60 patients with HUS, Stx or a Stx-producing E. coli could be detected in only .sup.23 % of fecal specimens whereas an IgM response to the O157 LPS was detected in 73% of these patients. In an epidemic situation, an immunoassay based on IgG responses to LPS was over 90% sensitive and specific for patients with recent culture-confirmed infection (39). Antibodies to the other antigen of the O157:H7 serotype, the H7 flagella, were not detected in any of the HUS patients studied by Chart et al. (40). In this same study, some patients showed responses to outer membrane proteins (OMPs) but this response was found to be due to contaminating LPS co-migrating with the OMPs.
Stx represents an obvious choice for an important antigen produced in vivo. However, numerous investigations of the serological response to Stx have yielded disappointing results. A curious phenomenon is that sera from most individuals without any history of infection with Stx-producing E. coli contain a substance that is capable of neutralizing Stx2. When ELISA rather than neutralization tests are conducted to detect antibodies against either Stx1 or Stx2, only a minority of HUS patients showed a response to these toxins (39, 41-43). Similar results are seen in patients infected with Stx-producing Shigella dysenteriae type (44) and may be related to the fact that Stx is cytotoxic to human B lymphocytes (45). Whatever the reason, these results have led experts to conclude that antitoxin response is not a useful tool in the serodiagnosis of Stx-producing E. Coli (39, 41, 42, 46)
Serological responses to E. coli O157:H7 and non-O157:H7 STEC in cattle have not been as extensively studied as in humans. Some cattle experimentally infected with E. coli O157 developed serum antibodies against Stx and O157 LPS but others did not (55). Detection of antibodies against Stx in cattle is not useful in predicting the potential for human disease since the prevalance of non-O157:H7 STEC of doubtful pathogenic significance is so high in cattle, as described above.
In summary, several years of research into the pathogenesis and immune response of E. coli O157:H7 infections have not yielded firm knowledge of the protective immune response or an ideal serodiagnostic tool. The O157 LPS is useful for serodiagnosis, but it is difficult to prepare and the response may be nonspecific since O157 LPS shares epitopes with E. coli O44 LPS (43) and the LPS of certain serogroups of Salmonella spp., Yersinia enterocolitica, Brucella abortus, and Vibrio cholerae non-O1 strains (reviewed in (47). It is also useless for detecting infection due to Stx-producing E. coli strains of non-O157 serogroups. On the other hand, Stx is clearly produced in vivo and is essential for disease, but is not useful for serodiagnosis.
Summary
Although extensive research has been conducted on the Shiga toxins expressed by this organism, there is very little known about other potential virulence factors of this pathogen. There is clearly a need in the art to have definitive diagnostic techniques available. We have discovered novel proteins that are secreted by Stx-producing strains of E. coli serotypes O157:H7 and O26:H11. These proteins are expressed during the course of infection and are highly immunogenic in humans, and is therefore useful as serodiagnostic tool. Furthermore, these proteins are also immunogenic in cattle. The invention teaches a method for determining cattle which are infected with EHEC. Cattle which test positive can be omitted from food production.